Major Depressive Disorder (MDD) is a frequently recurring disorder that is the second leading cause of disability worldwide and a significant risk factor for suicide. Despite better recognition and increased treatment, the prevalence of MDD and the overall rates of suicide remain unchanged. Although a wide variety of treatments are available, their overall efficacy remains modest, and only a third of treated patients show full remission. Novel treatments are urgently needed, yet the development of new types of antidepressants has been hindered by our limited understanding of the pathophysiology of MDD. In this proposal, we seek to perform the largest and most comprehensive molecular study of MDD in post-mortem brains to date. We will use the substantial post-mortem brain collection of the Lieber Institute for Brain Development to comprehensively study genome-wide expression and epigenetic signatures that are associated with MDD and suicide in the three brains regions of the corticolimbic circuit, consisting of the Anterior Cingulate Cortex (ACC), the Amygdala, and the Dorsal Lateral Prefrontal Cortex (DLPFC). We propose to perform RNA sequencing, DNA-methylation and chromatin accessibility (ATAC-seq) assays in the ACC and Amygdala of 400 post- mortem brain samples and to combine these results with of the DLPFC from Brainseq project. Our aims will then be to: (1) conduct case-control analyses of these two brain regions along with RNA-seq of the DLPFC to identify molecular signatures associated with MDD and integrate our findings across molecular profiles to provide additional mechanistic insights into the causes of MDD; (2) to take advantage of recent identification of genome-wide significant findings from GWAS studies in MDD and its closely related trait, neuroticism, to fine- map associated loci and identify expression quantitative trait loci (eQTLs) and changes in methylation and/or chromatin that may mediate the association between genetic marker and expression; (3) to perform a case- only analysis of the cases with MDD who died by suicide compared with cases with MDD who died by natural causes to identify molecular signatures and mechanisms associated with suicide; and, finally (4) to perform a replication of the main findings in an independent sample of 50 cases and 50 controls. In order to ensure the success of this proposal, we have assembled a strong interdisciplinary investigative team from the Johns Hopkins Mood Disorder research group and the Lieber Instituted for Brain development, with expertise in mood disorders, genomics, post-mortem brain studies, and statistical genetics. By investigating the functional genomics and epigenomics of MDD in a large and well characterized post-mortem brain collection, our study has strong potential to identify genes and networks that could be novel targets for a new generation of treatments.